Specific biomarker set for non-invasive diagnosis of liver cancer

ABSTRACT

Cells within liver tumor mass comprise a unique set of proteins/tumor antigens when compared to the normal liver tissues epithelial cells juxtaposed to the tumor. The presence of tumor antigens couples the production of auto-antibodies against these tumor antigens. The present invention relates to the identification and elucidation of a protein set that can act as a novel marker set for liver cancer diagnosis and prognosis. Specifically, it relates to a kit that enables diagnostic and prognostic measurement of auto-antibodies in serum of liver cancer patients. The present invention provides a non-invasive, specific, sensitive, and cost effective detection and quantification method by evaluating a set of validated liver cancer proteins/tumor antigens, which includes Bmi-1, VCC1, SUMO-4, RhoA, TXN, ET-1, UBE2C, HDGF2, FGF21, LECT2, SOD1, STMN4, Midkine, IL-17A or IL26, to complement the conventional diagnostic methods.

COPYRIGHT NOTICE/PERMISSION

A portion of the disclosure of this patent document contains materialwhich is subject to copyright protection. The copyright owner has noobjection to the facsimile reproduction by anyone of the patent documentor the patent disclosure as it appears in the Patent and TrademarkOffice patent file or records, but otherwise reserves all copyrightrights whatsoever. The following notice applies to the processes,experiments, and data as described below and in the drawings attachedhereto: Copyright © 2014, Vision Global Holdings Limited, All RightsReserved.

TECHNICAL FIELD

The present invention describes a detection and quantification methodfor a list of specific and novel Hepatocellular Carcinoma (HCC) tumorbiomarkers, by measuring the corresponding auto-antibodies in livercancer patients' sera. The set of biomarkers comprises Bmi1, VCC1,SUMO-4, RhoA, TXN, ET-1, UBE2C, HDGF2, FGF21, LECT2, SOD1, STMN4,Midkine, IL-17A and IL26. More specifically, this invention furtherdescribes a design of a high throughput and sensitive test kit readilyavailable to take patients' peripheral serum samples for detecting livercancers early and in a non-invasive manner by measuring theauto-antibodies against at least one of the biomarkers selected from thebiomarker set. The present invention further allows identification ofsignature biomarker patterns for staging, as well as the detection ofrecurrences during a monitoring period of post-chemotherapeutictreatment. The present invention would support automatic data analysis.

BACKGROUND OF INVENTION

Hepatocellular carcinoma (HCC) is the second most prevalent cancer inChina, which covers 5.7% of the total population [1]. Most HCC patientshave rapid tumor progressing resulting in high mortality rate. In orderto improve the overall survival, early diagnosis of the disease becomesessential. Currently, the most common way of detecting HCCs are bloodtests that measure level of HCC tumor markers such as alpha fetoprotein(AFP). AFP is a plasma protein produced by yolk sac and liver during thedevelopment of fetus serving as a form of serum albumin. In normalcondition, AFP level gradually decreases after birth and remain in lowlevel in adults. Increased level of tumor markers indicates probabilityof liver cancers. However, the major problem of the AFP test isexcessive false positive. It is because HCC is not the only cause forthe AFP level elevation, but alcoholic hepatitis, chronic hepatitis orcirrhosis also associates with increase of AFP.

Despite AFP test is commonly suggested for diagnosis of liver cancers,its result is not conclusive. Suspected patients will need to go throughultrasound imaging, CT scans or contrast MRI scans for furtherconfirmation. Liver biopsy will be taken to distinguish whether thetumor is benign or malignant. However, conventional detection of HCCscomes with several limitations: (a) About 20% of liver cancers does notproduce elevated level of the commonly used HCC tumor markers [2]. (b)Viral cirrhosis produces false positive results on the blood tests [3].(c) Ultrasound is not able to detect small tumors [4]. (d) CT scansrequire high radiation dose and are insensitive to tumors less than 1 cm[5]. (e) MRI scans are expensive and the procedure is time consuming.Due to these limitations, there are needs to develop novel biomarkersscreen with higher sensitivity and specificity for the purpose of earlydiagnosis of HCC and/or determining a prognosis of HCC to complement theconventional methods.

HCC tumor cells tend to produce a unique set of proteins when comparedto the normal liver epithelial cells juxtaposed to the tumor. Evaluationof validated HCC tumor biomarkers has great potential to facilitate thediagnosis of HCC. However, not all biomarkers themselves can be found inserum or urine for convenient diagnosis. Alternatively, theauto-antibodies which are specifically against the biomarkers provide anopportunity to evaluate the expression of the biomarkers. It has beendemonstrated in many cancers that the presence of tumor biomarkerscouples the production of auto-antibodies against these tumor antigens[6-8]. Detection on auto-antibodies in patients' sera would allow us toexamine the presence of biomarkers more efficiently. Ideally,examination of auto-antibodies from peripheral blood would be atestament for detecting liver cancers early, and in a non-invasivemanner. One common hurdle hindering clinical use of biomarkers is thatthey have not been validated after discovery. But once validated, suchtest would be cost effective and accurate. The design of the prototypealso supports high-throughput screening. This may alleviate the costrequired for conventional liver cancer diagnosis.

There follows a list of references that are occasionally cited in thespecification. Each of the disclosures of these references isincorporated by reference herein in its entirety.

-   [1] Chen J G, Zhang S W. Liver cancer epidemic in China: past,    present and future. Semin Cancer Biol. 2011; 21(1):59-69-   [2] Okuda K, Peters R L. Human alpha-1 fetoprotein. Hepatocellular    Carcinoma. 1976:353-67-   [3] Lok A S, Lai C L. Alpha-fetoprotein monitoring in Chinese    patients with chronic hepatitis E virus infection: role in the early    detection of hepatocellular carcinoma. Hepatology 1989; 9:110-115-   [4] Colombo M, de Franchis R, Del Ninno E, Sangiovanni A, De Fazio    C, Tommasini M, Donato M F, Piva A, Di Carlo V, Dioguardi N.    Hepatocellular carcinoma in Italian patients with cirrhosis. N Engl    J Med. 1991; 325:675-80-   [5] Sahani D V, Kalva S P. Imaging the Liver. The Oncologist. 2004;    9 (4): 385-397-   [6] Masutomi K, Kaneko S, Yasukawa M, Arai K, Murakami S,    Kobayashi K. Identification of serum anti-human telomerase reverse    transcriptase (hTERT) auto-antibodies during progression to    hepatocellular carcinoma. Oncogene. 2002 Aug. 29; 21(38):5946-50.-   [7] Karanikas V, Khalil S, Kerenidi T, Gourgoulianis K I, Germenis    A E. Anti-survivin antibody responses in lung cancer. Cancer Lett.    2009 Sep. 18; 282(2):159-66.-   [8] Wang Y Q, Zhang H H, Liu C L, Xia Q, Wu H, Yu X H, Kong W.    Correlation between auto-antibodies to survivin and MUC1 variable    number tandem repeats in colorectal cancer. Asian Pac J Cancer Prev.    2012; 13(11):5557-62.

SUMMARY OF INVENTION

In the present invention, a detection and quantification methodmeasuring the auto-antibodies against a list of specific tumor biomarkeraiming for diagnosing and staging cancers is provided. Comparing to thenormal liver epithelial cells, HCC tumor cells tend to produce a uniqueset of proteins. The evaluation of the unique protein set, biomarkers,will complement the conventional diagnostic methods and facilitate earlydetection of cancers.

By using a Two-Dimensional/Mass Spectrometry based method, a set ofliver cancer biomarkers from paired patients' biopsies (tumor biopsyversus juxtaposed normal tissue) is identified in the present inventioncomprising Bmi1, VCC1, SUMO-4, RhoA, TXN, ET-1, UBE2C, HDGF2, FGF21,LECT2, SOD1, STMN4, Midkine, IL-17A and IL26.

Specificity and accuracy of this set of liver cancer biomarkers are thenvalidated and taken together for diagnosis of liver cancers. In thepresent invention, proteins of the listed biomarkers are expressed fromcDNA clones, purified and coupled to fluorescent microsphere beads withdifferent emission wavelengths. Auto-antibodies present in patients'sera against the proteins immunologically bind to the protein-beadconjugate. The auto-antibodies subsequently interact with PE-conjugatedsecondary antibodies. The specific fluorescence signal of themicrosphere beads serves as an identifier for the conjugated biomarkers.By measuring the fluorescent intensity given by the PE-conjugatedsecondary antibodies at the complex, it allows the detection andquantification of the auto-antibodies. Since the auto-antibodies areproduced in the patients' sera in proportion to the abundance of thebiomarkers at HCC tumor cells, the higher fluorescent intensity resultedfrom higher concentration of auto-antibodies indicates the higherexpression of the corresponding biomarkers. The lowest detection limitof each biomarker to the total serum auto-antibodies is about 0.15ng/mL.

Comparing to sera from healthy subjects, the level of auto-antibodiesagainst the target biomarkers is at a higher concentration in cancerpatient. Moreover, comparing different sera from liver cancer patientsat different stages, signature patterns for staging may be generated.Thus, the present invention allows the non-invasive evaluation of thetargeted liver cancer biomarker. This enables the detection of HCC atearly stages and the identification of signature biomarker patterns forstaging, as well as the detection of recurrences during a monitoringperiod of post-chemotherapeutic treatment.

BRIEF DESCRIPTION OF THE DRAWINGS

Embodiments of the present invention are described in more detailhereinafter with reference to the drawings, in which:

FIG. 1 shows the difference in protein expression pattern between tumorbiopsy and juxtaposed normal tissue by two-dimensional/mass spectrometryleading to the identification of 15 specific biomarkers up-regulated inliver cancer; arrows indicate location of spots identified on a 2-D gelof the mass spectrometry.

FIG. 2 shows the set of 15 validated liver cancer biomarkers and theircorresponding molecular weight targeted and measured in the presentinvention.

FIG. 3 shows the workflow of expressing the biomarkers from cDNA clones.

FIG. 4 shows the workflow of purification of the biomarkers expressedfrom E. coli.

FIG. 5 shows the workflow of measuring the auto-antibodies by BioPlexsystem.

FIG. 6 shows the conjugation of biomarker protein to BioPlex bead.

FIG. 7 shows illustration of the complex of biomarker-BioPlex beadconjugate immunoreacting with primary antibody and PE-conjugatedsecondary antibody.

FIG. 8 shows the gel electrophoresis of the DNA insert released fromplasmid cut by restriction enzymes HindIII and BamH1.

FIG. 9 shows the Coomassie Blue stained SDS-PAGE verifying the IPTGinduction of (a) Bmi1, (b) SOD1, (c) IL-17A, (d) TXN and (e) Midkinebiomarkers.

FIG. 10 shows the elution profile of (a) Bmi1, (b) SOD-1 and (c) IL-17Ain AKTA.

FIG. 11 shows the Coomassie Blue stained SDS-PAGE verifying thepurification of His-tagged (a) Bmi1, (b) SOD-1 and (c) IL-17Abiomarkers; Fraction A is bacteria without IPTG induction; Fraction B isbacteria with IPTG induction; Fraction C is bacterial lysate.

FIG. 12 shows the standard curve showing the fluorescence intensityagainst the concentration of anti-Bmi1 antibody.

FIG. 13 is a schematic diagram showing the design of the test: Patientserum containing auto-antibodies are mixed to a well containing 15 typesof beads corresponding to the 15 biomarkers of the biomarker set,followed by the addition of PE-conjugated secondary antibody.

DEFINITIONS

The term “biomarker” refers to the protein uniquely expressed orup-regulated in the tumor comparing to the normal epithelial cells.

The term “biomarker set” refers to the specific combination of thebiomarkers identified from paired patients' biopsies (tumor biopsyversus juxtaposed normal tissue) and is the target of the measurement inthe present invention.

The term “auto-antibodies” refers to the anti-bodies produced by thepatient body coupling to the expression of the tumor biomarker and it ispresent in the circulation and can be collected in the peripheral serum.

Bmi1 (Polycomb Ring Finger) is a protein component of a Polycomb Group(PcG) multiprotein PRC1-like complex. It is responsible for maintainingthe transcriptionally repressive state of many genes, including Hoxgenes, throughout development. The regulation is via monoubiquitinationof histone H2A ‘Lys-119’, which modifies histone and remodels chromatin,rendering the expression.

VCC1 or CXCL17 (Chemokine (C-X-C Motif) Ligand 17) has an essential rolein angiogenesis and possibly in the development of tumors. It is alsosuggested that it is a housekeeping chemokine regulating the recruitmentof non-activated blood monocytes and immature dendritic cells intotissues. It may also play a role in the innate defense againstinfections. Malfunction of VCC1 is associated with duodenitis andcholera.

SUMO-4 (Small Ubiquitin-Like Modifier 4) belongs to the family of smallubiquitin-related modifiers and located in the cytoplasm. It covalentlyattaches to the target protein, IKBA, in order to control itssubcellular localization, stability, or activity. This eventually leadsto a negative regulation of NF-kappa-B-dependent transcription of theIL12B gene.

RhoA (Ras Homolog Family Member A) regulates the signaling pathwaylinking plasma membrane receptors to the assembly of focal adhesions andactin stress fibers. It also involves in microtubule-dependent signalingessential during cell cycle cytokinesis, and other signaling pathwaysinvolved in stabilization of microtubules and cell migrations andadhesion.

TXN (Thioredoxin) forms homodimer and is involved in redox reactionsthrough the reversible oxidation of its active center dithiol to adisulfide and catalyzes dithiol-disulfide exchange reactions. It hasbeen reported to be associated with breast mucinous carcinoma.

ET-1 (Endothelin 1) is a potent vasoconstrictor produced by vascularendothelial cells. It binds to endothelin receptors widely expressed inall tissues, including non-vascular structure like epithelial cells,glia, and neurons. Apart from the main role in maintenance of vasculartone, it is also suggested to have co-mitogenic activity and potentiatethe effects of other growth factors.

UBE2C (Ubiquitin-Conjugating Enzyme E2C) belongs to the family of E2ubiquitin-conjugating enzyme. This is one of the three enzymes involvedin ubiquitination, which is an important cellular mechanism fortargeting abnormal proteins for degradation. More specifically, UBE2C isrequired for the targeted degradation of mitotic cyclins and for cellcycle progression. Thus, it is believed that this protein may be alsoinvolved in cancer progression.

HDGF2 is called hepatoma-derived growth factor 2. This protein which ishighly expressed in a variety of tumors has been reported to play apivotal role in the development and progression of several tumors.Although the mechanism is yet to be identified, it is suggested thatHDGF2 has mitogenic, angiogenic, neurotrophic and antiapoptoticactivity.

FGF21 (Fibroblast Growth Factor 21) is a family member of the FGF familywhich is involved in vary biological processes including embryonicdevelopment, cell growth, morphogenesis, tissue repair, tumor growth andinvasion. More specifically, FGF21 stimulates glucose update indifferentiated adipocytes via the induction of glucose transporterSLC2A1/GLUT1 expression. It has been found that FGF21 is associated withfatty liver disease.

LECT2 (Leukocyte Cell Derived Chemotaxin 1) is a secretory protein actsas a chemotactic factor to neutrophils and stimulates the growth ofchondrocytes and osteoblasts. This protein is associated with acuteliver failure.

SOD1 (Superoxide Dismutase 1) is a Cu/Zn-containing antioxidant enzymeresponsible for destroying free superoxide radicals into molecularoxygen and hydrogen peroxide in the cytosol, the nucleus, and theintermembrane space of the mitochondria. It is important for maintaininglow levels of superoxide in the cytosol, thus protecting the cell fromoxidative stress and subsequent cell death.

STMN4 (Stathmin-Like 4) is a small regulatory protein which is believedto have a role in relaying integrating diverse intracellular signalingpathways, which in turn, controls cell proliferation, differentiationand functions. It is also shown that this protein contributes to thecontrol of microtubule dynamics by inhibiting the polymerization ofmicrotubules and/or favoring their depolymerization.

Midkine or NEGF2 (Neurite Growth-Promoting Factor 2) is a secretorygrowth factor that binds heparin and responsive to retinoic acid.Midkine promotes cell growth, migration and angiogenesis, in particularduring tumorigenesis. It has already been demonstrated to be associatedwith breast adenocarcinoma and soft tissue sarcoma.

IL-17A (Interleukin 17A) is a proinflammatory cytokine produced by theactivated T cells. It regulates the activity of NF-kappaB andmitogen-activated protein kinases, stimulates the expression of IL6 andcyclooxygenase-2, and enhances the production of nitric oxide. Severalchronic inflammation and sclerosis are usually associated with IL-17Aelevation.

IL-26 (Interleukin 26) belongs to the IL-10 cytokine family and isproduced by the activated T cells and targets epithelial cells forsignal transduction. It binds strongly to glycosaminoglycans such asheparin, heparan sulphate, and dermatan sulfate on cellular surfaceswhich act similarly to coreceptors in order to enrich IL-26 on thesurface of producer and target cells.

DETAILED DESCRIPTION OF INVENTION

In the following description, the biomarker/biomarkers, thecorresponding embodiments of thedetection/validation/identification/quantification methods are set forthas preferred examples. It will be apparent to those skilled in the artthat modifications, including additions and/or substitutions, may bemade without departing from the scope and spirit of the invention.Specific details may be omitted so as not to obscure the invention;however, the disclosure is written to enable one skilled in the art topractice the teachings herein without undue experimentation.

In the present invention, the set of liver tumor biomarkers fordetection and quantification of liver cancer is first identified bytwo-dimensional/mass spectrometry resolving the difference in thepattern of proteins expression between the paired patients' biopsies(tumor biopsy versus juxtaposed normal tissue) (FIG. 1). The biomarkersare validated by immunohistochemical staining on paraffin-sectioned HCCblocks, and Western Blotting in HCC patients' sera. This results in afinalized list of 15 biomarkers to be evaluated in the present inventionfor the liver cancer diagnosis purpose (FIG. 2).

Based on the amino acid sequences of the targeted biomarkers,commercially synthesized cDNA clones are employed for the expression ofthe biomarker set (FIG. 3). Proteins expressed from the cDNA clones arethen subjected to a series of steps of purifications (FIG. 4). Thepurified biomarkers are subsequently conjugated via stable amide bondswith BioPlex beads (FIGS. 5, 6), a type of fluorescent microsphere beadsand available in a panel which give unique fluorescent signalsindividually for identification at a multiplex set up. The biomarkers onthe beads are recognized by the specific primary antibodies, which aresubsequently bound by an anti-human secondary antibody conjugated withPE (FIG. 7). Thus the BioPlex machine simultaneously measures twosignals from the complex. The fluorescence given by the BioPlex beadsserves as an identifier, while the signal from the PE indicates thepresence of the biomarker in the complex. This also helpsdifferentiating the biomarker-bead conjugates bound by the anti-bodycascade from those with no immuno-reactivity with antibodies.

To prove the significance of the biomarkers in the present invention,the cDNA clones are confirmed by restriction enzyme cut (FIG. 8). Thetransformed bacteria is induced by IPTG to express the biomarkerproteins. The protein expression verified by SDS-PAGE and Coomassie Bluestaining reveals the protein bands (FIG. 9 a-e). The His-tagged Bmi1,SOD1 and IL-17A proteins are purified by AKTA (FIG. 10 a-c) and thenverified by SDS-PAGE and Coomassie Blue staining (FIG. 11 a-c).

Sensitivity of the test is measured by spiking in a serial dilution ofthe antibodies. The lowest concentration of the antibody added that cangive signal suggests the sensitivity of that particular biomarker.Meanwhile a standard curve is constructed showing the fluorescenceintensity of the PE against the serial dilutions of the antibodies (FIG.12). The standard curve will be used for estimating the concentration ofthe biomarker specific auto-antibodies in the patient sera by comparingthe PE intensity.

In the present invention, a multiplex of 15 different Bioplex beadsindividually giving unique fluorescence are conjugated with thebiomarker set and preloaded in the wells of a plate (FIG. 13). To awell, patient serum containing auto-antibodies is loaded and allowed tointeract with the biomarker conjugates. The PE-conjugated secondaryantibodies are then added and bind to the auto-antibodies. In themachine, the excess secondary antibodies are washed away, the complexcomprising the biomarker-bead conjugate and cascade of antibodies aremeasured individually. The unique fluorescence signal of the Bioplexbead identifies the biomarkers, while the PE signal from the samecomplex indicates the presence of the auto-antibodies as the primaryantibody (FIG. 7). Taken together, the measurement will suggest thepresence of auto-antibodies and the relative concentration in thepresents' sera.

In a standard randomized trial design, the mean of the relative level ofauto-antibodies between the healthy group and patients diagnosed withliver cancer is compared. Student T test is used to analyze thevariation significance. The significant difference indicates that thebiomarker is specific for liver cancer. After the verification trials,ranges of the concentration of biomarker specific auto-antibodies willbe obtained for the liver cancer positive and negative patients andserve as reference point for the future diagnosis. Meanwhile, expressionpattern of the auto-antibodies is also compared between liver cancerpatients of different stages. The signature patterns of the biomarkerexpressions will indicate the HCC staging.

Taken together, the measurement of the relative auto-antibodies leveland the expression pattern of the biomarkers, the present inventionrepresents a different avenue to complement conventional liver cancerdiagnosis. The present invention further enables non-invasive detectionof auto-antibodies against the validated targets in patients' sera ofthe present invention, identifying the extent and the characteristics ofthe disease. Apart from early detection for stage I liver cancers, thepresent invention also enables the generation of signature patterns forstaging, and the detection of recurrences during a monitoring period ofpost-mastectomy or post-chemotherapeutic treatment.

EXAMPLES

The following examples are provided by way of describing specificembodiments of this invention without intending to limit the scope ofthis invention in any way.

Example 1a Protein Extraction from Patients' Biopsies

500 mg of the paired patients' biopsies (tumor biopsy versus juxtaposednormal tissue) are collected and washed with PBS. The tissues are frozenby submerging into liquid nitrogen and immediately homogenized withpestle and mortar. To the homogenized samples, lysis solution (8M Urea,4% CHAPS, 2% IPG Buffer, 0.2 mg/ml PMSF) is added, then vortex for atleast 5 min until the tissues are completely dispersed. The lysates arethen clarified by centrifugation at 14,000 rpm for 10 minutes at 4° C.The supernatants are further cleaned up by 2D Clean Up kit (Amersham) toremove the salt and impurities. The pellets are resuspended with minimumvolume of Rehydration Solution (No DTT & IPG Buffer added). The proteinconcentrations are then measured by Bio-Rad protein assay and aliquotsof 200 g/per tube are stored at −70° C.

Example 1b Resolving Proteins by Two-Dimensional Electrophoresis

To 1 ml rehydration stock solution, 2.8 mg DTT, 5 μl pharmalyte or IPGBuffer, and 2 μl bromophenol blue are added. 50-100 μg of protein sampleis added to the 13 cm Immobiline DryStrip (IPG strip) containing 250 μlof rehydration solution. After removing the protective cover, the IPGstrip is positioned in the strip holder with the gel side facing down,and overlaid with Cover Fluid to prevent dehydration duringelectrophoresis. The strip is then placed on to Ettan IPGphor (Amersham)for isoelectric focusing (first dimensional electrophoresis).

After the first-dimensional electrophoresis, the IPG strip isequilibrated with equilibrate solution (6 M Urea 2% SDS, 50 mM Tris HClpH 6.8, 30% Glycerol, 0.002% Bromophenol blue, 100 mg DTT per 10 mlbuffer and 250 mg IAA per 10 ml buffer), and then washed with 1×SDSrunning Buffer for 4-5 times. The IPG strip is placed on top of thesecond-dimension gel and overlaid with sealing solution (0.5% LowMelting agarose, 0.002% Bromophenol Blue in 1×SDS running Buffer). Thesecond-dimensional electrophoresis is then carried out at 30 mA forfirst 15 min followed by 60 mA for 3-4 h.

Upon the completion of the second dimensional electrophoresis, the gelis removed from the cassette, fixed and stained with silver nitrate. 15spots representing 15 up-regulated proteins are identified (FIG. 1). Toidentify the proteins (FIG. 2), the silver stained gel slices aredestained and trypsinized to release the protein from the gel forMALDI-TOF analysis.

Example 2a SEQ ID NO.1 Amino Acid Sequence of Bmi1

MHRTTRIKITELNPHLMCVLCGGYFIDATTIIECLHSFCKTCIVRYLETSKYCPICDVQVHKTRPLLNIRSDKTLQDIVYKLVPGLFKNEMKRRRDFYAAHPSADAANGSNEDRGEVADEDKRIITDDEIISLSIEFFDQNRLDRKVNKDKEKSKEEVNDKRYLRCPAAMTVMHLRKFLRSKMDIPNTFQIDVMYEEEPLKDYYTLMDIAYIYTWRRNGPLPLKYRVRPTCKRMKISHQRDGLTNAGELESDSGSDKANSPAGGIPSTSSCLPSPSTPVQSPHPQFPHISSTMNGTSNSPSGNHQSSFANRPRKSSVNGSSATSSG

Example 2b SEQ ID NO.2 Amino Acid Sequence of VCC1

MKVLISSLLLLLPLMLMSMVSSSLNPGVARGHRDRGQASRRWLQEGGQECECKDWFLRAPRRKFMTVSGLPKKQCPCDHFKGNVKKTRHQRHHRKPNKHS RACQQFLKQCQLRSFALPL

Example 2c SEQ ID NO.3 Amino Acid Sequence of SUMO-4

MANEKPTEEVKTENNNHINLKVAGQDGSVVQFKIKRQTPLSKLMKAYCEPRGLSVKQIRFRFGGQPISGTDKPAQLEMEDEDTIDVFQQPTGGVY

Example 2d SEQ ID NO.4 Amino Acid Sequence of RhoA

MAAIRKKLVIVGDGACGKTCLLIVFSKDQFPEVYVPTVFENYVADIEVDGKQVELALWDTAGQEDYDRLRPLSYPDTDVILMCFSIDSPDSLENIPEKWTPEVKHFCPNVPIILVGNKKDLRNDEHTRRELAKMKQEPVKPEEGRDMANRIGAFGYMECSAKTKDGVREVFEMATRAALQARRGKKKSGCLVL

Example 2e SEQ ID NO.5 Amino Acid Sequence of TXN

MVKQIESKTAFQEALDAAGDKLVVVDFSATWCGPCKMIKPFFHSLSEKYSNVIFLEVDVDDCQDVASECEVKCMPTFQFFKKGQKVGEFSGANKEKLEAT INELV

Example 2f SEQ ID NO.6 Amino Acid Sequence of ET-1

MDYLLMIFSLLFVACQGAPETAVLGAELSAVGENGGEKPTPSPPWRLRRSKRCSCSSLMDKECVYFCHLDIIWVNTPEHVVPYGLGSPRSKRALENLLPTKATDRENRCQCASQKDKKCWNFCQAGKELRAEDIMEKDWNNHKKGKDCSKLGKKCIYQQLVRGRKIRRSSEEHLRQTRSETMRNSVKSSFHDPKLKGNPS RERYVTHNRAHW

Example 2g SEQ ID NO.7 Amino Acid Sequence of UBE2C

MASQNRDPAATSVAAARKGAEPSGGAARGPVGKRLQQELMTLMMSGDKGISAFPESDNLFKWVGTIHGAAGTVYEDLRYKLSLEFPSGYPYNAPTVKFLTPCYHPNVDTQGNICLDILKEKWSALYDVRTILLSIQSLLGEPNIDSPLNTHAAELWKNPTAFKKYLQETYSKQVTSQEP

Example 2h SEQ ID NO.8 Amino Acid Sequence of HDGF2

MARPRPREYKAGDLVFAKMKGYPHWPARIDELPEGAVKPPANKYPIFFFGTHETAFLGPKDLFPYKEYKDKFGKSNKRKGFNEGLWEIENNPGVKFTGYQAIQQQSSSETEGEGGNTADASSEEEGDRVEEDGKGKRKNEKAGSKRKKSYTSKKSSKQSRKSPGDEDDKDCKEEENKSSSEGGDAGNDTRNTTSDLQKTS EGT

Example 2i SEQ ID NO.9 Amino Acid Sequence of FGF21

MDSDETGFEHSGLWVSVLAGLLLGACQAHPIPDSSPLLQFGGQVRQRYLYTDDAQQTEAHLEIREDGTVGGAADQSPESLLQLKALKPGVIQILGVKTSRFLCQRPDGALYGSLHFDPEACSFRELLLEDGYNVYQSEAHGLPLHLPGNKSPHRDPAPRGPARFLPLPGLPPALPEPPGILAPQPPDVGSSDPLSMVGPS QGRSPSYAS

Example 2j SEQ ID NO.10 Amino Acid Sequence of LECT2

MFSTKALLLAGLISTALAGPWANICAGKSSNEIRTCDRHGCGQYSAQRSQRPHQGVDVLCSAGSTVYAPFTGMIVGQEKPYQNKNAINNGVRISGRGFCVKMFYIKPIKYKGPIKKGEKLGTLLPLQKVYPGIQSHVHIENCDSSDPTAY L

Example 2k SEQ ID NO.11 Amino Acid Sequence of SOD1

MATKAVCVLKGDGPVQGIINFEQKESNGPVKVWGSIKGLTEGLHGFHVHEFGDNTAGCTSAGPHFNPLSRKHGGPKDEERHVGDLGNVTADKDGVADVSIEDVISLSGDHCIIGRTLVVHEKADDLGKGGNEESTKTGNAGSRLACGVIG IAQ

Example 21 SEQ ID NO.12 Amino Acid Sequence of STMN4

MTLAAYKEKMKELPLVSLFCSCFLADPLNKSSYKYEADTVDLNWCVISDMEVIELNKCTSGQSFEVILKPPSFDGVPEFNASLPRRRDPSLEEIQKKLEAAEERRKYQEAELLKHLAEKREHEREVIQKAIEENNNFIKMAKEKLAQKMESNKENREAHLAAMLERLQEKDKHAEEVRKNKELKEEASR

Example 2m SEQ ID NO.13 Amino Acid Sequence of Midkine

MQHRGFLLLTLLALLALTSAVAKKKDKVKKGGPGSECAEWAWGPCTPSSKDCGVGFREGTCGAQTQRIRCRVPCNWKKEFGADCKYKFENWGACDGGTGTKVRQGTLKKARYNAQCQETIRVTKPCTPKTKAKAKAKKGKGKD

Example 2n SEQ ID NO.14 Amino Acid Sequence of IL-17A

MTPGKTSLVSLLLLLSLEAIVKAGITIPRNPGCPNSEDKNFPRTVMVNLNIHNRNTNTNPKRSSDYYNRSTSPWNLHRNEDPERYPSVIWEAKCRHLGCINADGNVDYHMNSVPIQQEILVLRREPPHCPNSFRLEKILVSVGCTCVTPI VHHVA

Example 2o SEQ ID NO.15 Amino Acid Sequence of IL-26

MLVNFILRCGLLLVTLSLAIAKHKQSSFTKSCYPRGTLSQAVDALYIKAAWLKATIPEDRIKNIRLLKKKTKKQFMKNCQFQEQLLSFFMEDVFGQLQLQGCKKIRFVEDFHSLRQKLSHCISCASSAREMKSITRMKRIFYRIGNKGIY KAISELDILLSWIKKLLESSQ

Example 3a Expression of Biomarker Set

His tagged plasmids containing cDNA inserts encoding the biomarker setis transformed into DH5 competent cells (301, FIG. 3). Single colony ispicked and allowed to grow in bacterial culture (302). The number ofplasmid is expanded and extracted from the bacteria by miniprep. Theplasmid is further transformed into BL21DE3 or BL21DE3pLysS competentcells. Transformed bacteria are selected and grew in 2×100 ml LB medium.When the bacterial culture reaches the optical density of 0.06, 200 μMof IPTG is added to 100 ml bacterial culture (303). Another 100 ml ofbacterial culture without IPTG is used as negative control. Thebacterial cultures are incubated at 30° C. with shaking 500 μl of thebacterial cultures are saved and stored at −20° C. 3 h after theincubation and in the next morning after incubating overnight.

Bacterial cultures with and without IPTG induction are mixed together ina 500 ml centrifuge bottle. Bacterial cells are collected bycentrifugation at 9000 rpm for 20 min at 4° C. (304). 500 μl ofsupernatant is saved as another negative control and the remainingsupernatant is discarded. The bacterial cultures and negative controlscollected in different points are run on a SDS-PAGE to resolve theprotein (305). The gel is then stained with Coomassie Blue overnight.After destaining the gel, the protein induction can be confirmed bychecking the size and comparing with the negative controls.

Example 3b Protein Purification for Biomarker Set

The bacterial cell pellets are resuspended in 10 ml solubilizationbuffer by vortex at room temperature. Keeping the resuspended cells in50 ml centrifuge tube on ice, the cells are completely lysed bysonication at amplitude 70% 10 rounds of 30 s with interval of 30 s(401, FIG. 4). The lysed cells are centrifuged at 10,000 rpm for 1 h at4° C. (402). Supernatants are transferred into dialysis tubing andsubmerged in 1 L unfiltered starting buffer for 4-6 h at 4° C. withconstant stirring (403). Dialysis is continued with another 1 L startingbuffer overnight. The supernatant is further filtered with 0.22 μmfilter disc and syringe. To the AKTA machine equipped with 0.1M Nickelsulfate charged HiTrap chelating column (404), filtered samples areloaded (405). A program is set at the AKTA machine that the eluent iscollected in fractions automatically (406). Proteins purified fromdifferent fractions are checked by SDS-PAGE analysis (407).

Example 4a Protein Coupling with Bio-Plex Beads

The purified proteins of the biomarker set are coupled with Bio-Plexbeads (Bio-Rad) (501) according to the manufacturer's manual. In brief,uncoupled bead is vortexed for 30 s and then sonicated for 15 s.1,250,000 beads are collected in a reaction tube by centrifugation of100 μl bead at maximum speed for 4 min. After washing with 100 μl beadwash buffer by centrifugation, the beads are resuspended in 80 μl beadactivation buffer. To the beads 10 μl 50 mg/ml freshly prepared S-NHSand 10 μl 50 mg/ml freshly prepared EDAC are added, followed by 20 minincubation in dark at room temperature (FIG. 6). The beads are thenwashed with 150 μl PBS twice.

To the washed beads, 10 μg proteins are added and the total volume istopped up with PBS to 500 μl, and allowed to incubate for 2 h withshaking in dark. Supernatant is removed after centrifugation at maximumspeed for 4 min. 250 μl blocking buffer is added to the beads and shookin dark for 30 min, followed by centrifugation at maximum speed for 4min and removal of supernatant. The beads are briefly washed and thenresuspended in the storage buffer for storage at 4° C. The numbers ofthe beads are counted with a hemocytometer.

Example 4b Validation of Protein-Bead Coupling

To a HTS 96 well plate, 50 μl of conjugated Bio-Plex beads (100beads/μl) is added to react with primary followed by secondaryantibodies (502). A serial dilution of the commercially availableprimary anti-bodies against the biomarker set is prepared as 8,000,4,000, 1,000, 250, 62.5, 15.625, 3.906, 0.977, 0.244 and 0.061 ng/ml. 50μl of each dilution is added to each well. Two negative controls areperformed by excluding the primary antibodies, and both primary andsecondary antibodies in the wells. The plate is then sealed with a foiland kept on a shaker for 30 min at 350 rpm, avoiding exposure to light.

After incubation, the beads are washed three times with 150 μl PBS. 50μl of PE-conjugated secondary antibody (8,000 ng/ml) is added into eachwell except negative controls. The plate is sealed again and incubatedin dark for 30 min with shaking. Excess antibodies are then washed awayby PBS. The Bio-Plex machine is calibrated with the calibration kit andvalidation kit. After the HTS plate is loaded to the machine, signalsfrom both the Bio-Plex beads and the PE conjugated at the secondaryantibodies (503) are measured (schematic diagram is shown in FIG. 7). Acalibration curve is generated by Logistic-5PL.

Example 4c Collection of Serum Samples and Measurement ofAuto-Antibodies by BioPlex System

Whole-blood samples are clotted by standing at 37° C. for 1 h. Seracontaining the auto-antibodies is collected at the supernatant aftercentrifugation at 1000 g room temperature for 10 min. The serum samplesare diluted with PBS when necessary. To a HTS plate preloaded withBioplex beads conjugated with biomarker set, the serum samples areloaded and incubated for 30 min with shaking (FIG. 13). Similar to thesteps described in Example 4b, to the PBS washed beads, 50 μl ofPE-conjugated secondary antibody (8000 ng/ml) is added, followed byshaking for another 30 min. After three rounds of washing, the plate isloaded to the Bio-Plex machine and the fluorescence signal is measured(504). The concentration of the auto-antibodies can then be calculatedfrom the standard curves.

The foregoing description of the present invention has been provided forthe purposes of illustration and description. It is not intended to beexhaustive or to limit the invention to the precise forms disclosed.Many modifications and variations will be apparent to the practitionerskilled in the art.

The embodiments are chosen and described in order to best explain theprinciples of the invention and its practical application, therebyenabling others skilled in the art to understand the invention forvarious embodiments and with various modifications that are suited tothe particular use contemplated. It is intended that the scope of theinvention be defined by the following claims and their equivalence.

INDUSTRIAL APPLICABILITY

The presently claimed method and kit comprising the 15 identifiedbiomarkers can not only be used to identify and quantify the presence ofauto-antibodies in the patents' sera in order to detect and/or stage theliver cancer, but are also useful in drug development targeting thesemarkers for specifically treating the liver cancer.

What is claimed is:
 1. A method for measuring the presence ofhepatocellular carcinoma (HCC) biomarkers in a subject suspected ofhaving HCC, the method comprising: a. obtaining a serum sample from thesubject suspected of having HCC and measuring the serum for the presenceof primary auto-antibodies (primary biomarker auto antibodies) against aset of biomarkers, wherein the set of biomarkers comprises Bmi-1, VCC1,SUMO-4, RhoA, TXN, ET-1, UBE2C, HDGF2, FGF21, LECT2, SOD1, STMN4,Midkine, IL-17A and IL26; b. detecting the presence of the HCCbiomarkers in the subject suspected of having HCC, the method comprisingthe steps of: i. mixing the serum sample with a set of biomarkerconjugates to allow the primary biomarker auto antibodies, if present inthe serum sample, to bind to the set of biomarker conjugates and washingaway any unbound antibodies; wherein the set of biomarker conjugatescomprises each of the biomarkers in the set of biomarkers conjugated viaan amide bond to a unique fluorescent microsphere bead, wherein eachunique fluorescent microsphere bead associated with a specificparticular biomarker in the set of biomarkers has a different emissionwavelength for each biomarker, wherein the biomarker conjugates arecapable of being bound by a specific primary biomarker auto antibodypresent in the subject's serum sample, ii. adding to the mixture formedin step i. anti-human secondary antibodies conjugated with phycoerythrin(PE), which are capable of binding primary biomarker auto antibodies;and allowing the anti-human secondary antibodies conjugated with PE tobind to specific primary antibodies bound to biomarker conjugates toform a fluorescent bead-biomarker-auto antibody-PE conjugated antibodycascade, and washing away an unbound antibodies; and iii. measuring themixture formed in step ii. for the presence fluorescentbead-biomarker-auto antibody-PE conjugated antibody cascade to determinewhether the subject's serum contained primary biomarker auto antibodies.2. The method of claim 1 wherein the unique fluorescent signal from themicrosphere beads serves to identify which biomarker in the set ofbiomarkers is present and wherein the signal from the PE indicates thepresence of the biomarker conjugate.
 3. The method of claim 2, whereinfluorescent intensity given by the PE-conjugated secondary antibodies inthe fluorescent bead-biomarker-auto antibody-PE conjugated antibodycascade is measured to allow the detection and quantification of theprimary biomarker auto antibodies.
 4. The method of claim 1, wherein theset of biomarkers consists of Bmi-1, VCC1, SUMO-4, RhoA, TXN, ET-1,UBE2C, HDGF2, FGF21, LECT2, SOD1, STMN4, Midkine, IL-17A and IL26.
 5. Amethod for measuring the presence of hepatocellular carcinoma (HCC)biomarkers in a plurality of subjects having HCC at different stages,the method comprising: a. obtaining a serum sample from a plurality ofsubjects having HCC at different stages and measuring the serum for thepresence of primary auto-antibodies (primary biomarker auto antibodies)against a set of biomarkers, wherein the set of biomarkers comprisesBmi-1, VCC1, SUMO-4, RhoA, TXN, ET-1, UBE2C, HDGF2, FGF21, LECT2, SOD1,STMN4, Midkine, IL-17A and IL26; b. detecting the presence of the HCCbiomarkers in the plurality of subjects having HCC at different stages,the method comprising the steps of: i. mixing the serum sample with aset of biomarker conjugates to allow the primary biomarker autoantibodies, if present in the serum sample, to bind to the set ofbiomarker conjugates and washing away any unbound antibodies; whereinthe set of biomarker conjugates comprises each of the biomarkers in theset of biomarkers conjugated via an amide bond to a unique fluorescentmicrosphere bead, wherein each unique fluorescent microsphere beadassociated with a specific particular biomarker in the set of biomarkershas a different emission wavelength for each biomarker, wherein thebiomarker conjugates are capable of being bound by a specific primarybiomarker auto antibody present in the subject's serum sample, ii.adding to the mixture formed in step i. anti-human secondary antibodiesconjugated with phycoerythrin (PE), which are capable of binding primarybiomarker auto antibodies; and allowing the anti-human secondaryantibodies conjugated with PE to bind to specific primary antibodiesbound to biomarker conjugates to form fluorescent bead-biomarker-autoantibody-PE conjugated antibody cascade, and washing away an unboundantibodies; and iii. measuring the mixture formed in step ii. for thepresence fluorescent bead-biomarker-auto antibody-PE conjugated antibodycascade to determine whether the plurality of subjects' serum containedprimary biomarker auto antibodies.